Abstract:

The present invention relates to novel antibodies and their use for
detecting, imaging, staging, treating and monitoring of prostate cancer,
and/or metastasis thereof. The present invention also relates to novel
pharmaceutical compositions for the treatment of prostate cancer.
Furthermore the present invention relates to assay systems and kits for
detecting, imaging, staging, treating and monitoring of prostate cancer,
and/or metastasis thereof.

63. The isolated antibody or antigen binding fragment thereof according to
claim 62, wherein the antigen binding fragment is selected from the group
consisting of a Fab fragment, a F(ab')2 fragment, and a Fv fragment.

64. A pharmaceutical composition for targeted treatment of prostate cancer
expressing a PSMA polypeptide comprising SEQ ID NO.:22 and/or metastases
thereof, the pharmaceutical composition comprising the antibody or
antigen binding fragment thereof according to claim 62, linked to a
cytotoxic drug and a pharmaceutically acceptable carrier, wherein said
antibody is available for targeted binding to said PSMA polypeptide and
said linked cytotoxic drug remains biologically active.

65. The pharmaceutical composition according to claim 64, wherein the at
least one cytotoxic drug is selected from the group consisting of
Iodine-125, Iodine-131, cyclophosphamide, Yttrium-90, paclitaxel,
IFN-alpha, and IL-2.

66. The pharmaceutical composition according to claim 65, wherein said
antibody is a monoclonal antibody.

67. A composition for detection of prostate cancer expressing a PMSA
polypeptide comprising SEQ ID NO.:22 and/or metastases thereof in an
individual and/or in a sample obtained therefrom, which comprises the
antibody or antigen binding fragment thereof according to claim 62
adapted to be linked to a detectable label in association with a
physiologically acceptable carrier or an in vitro acceptable carrier,
wherein said antibody is available for binding and said detectable label
when linked to said antibody remains detectable.

68. The composition according to claim 67, wherein said detectable label
is selected from the group consisting of a radioactive label, a
fluorescent label, a nuclear magnetic resonance active label, a
luminescent label, a chromophore label, a positron emitting isotope for
PET scanner, a chemiluminescence label and an enzymatic label.

70. The isolated antibody or antigen binding fragment thereof, according
to claim 69, wherein the antibody is a monoclonal antibody or a
polyclonal antibody.

71. The isolated antibody or antigen binding fragment thereof, according
to claim 69, wherein the antigen binding fragment is selected from the
group consisting of a Fab fragment, a F(ab')2 fragment and a Fv
fragment.

72. A pharmaceutical composition comprising the antibody of claim 69, and
a pharmaceutically acceptable carrier.

73. The pharmaceutical composition of claim 72, wherein the antibody is
conjugated to a cytotoxic drug.

74. The pharmaceutical composition of claim 73, wherein the cytotoxic drug
is selected from the group consisting of Iodine-125, Iodine-131,
cyclophosphamide, Yttrium-90, paclitaxel, IFN-alpha and IL-2.

75. The pharmaceutical composition of claim 72, wherein the antibody is
conjugated to a detectable label.

76. The pharmaceutical composition of claim 75, wherein the detectable
label is selected from the group consisting of a radioactive label, a
fluorescent label, a nuclear magnetic resonance active label, a
luminescent label, a chromophore label, a positron emitting isotope for
PET scanner, a chemiluminescence label and an enzymatic label.

Description:

BACKGROUND OF THE INVENTION

[0001](a) Field of the Invention

[0002]The invention relates to novel antibodies and their use for
detecting, imaging, staging, treating and monitoring of prostate cancer,
and/or metastasis thereof. Furthermore, the invention also relates to
novel pharmaceutical compositions for the treatment of prostate cancer.

[0005]Prostate cancer is the second most frequently diagnosed cancer in
Canadian and American men, after non-melanoma skin cancer, which is
rarely fatal. More importantly, after lung cancer, prostate cancer is the
most common cause of cancer-related death. The risk of developing
prostate cancer increases significantly with age, particularly for men
over 50. For men under 50 years of age the disease is uncommon and death
from it is rare.

[0006]Prostate cancer accounts for an estimated 28% of newly diagnosed
cancer in Canadian men and more than 12% of cancer-related deaths. The
current lifetime risk of a Canadian man being diagnosed with prostate
cancer is about 1 in 8. In the United States, prostate cancer accounts
for approximately 32% of male cancer diagnoses and 14% of cancer deaths.
Studies in the United States suggest that the incidence rate may be
approaching 1 in 6 men.

[0007]Because the incidence of prostate cancer increases with age, it is
clear that the burden of this illness will increase dramatically in the
coming decades. The aging of the population, particularly the baby
boomers, will have important long-term implications for the number of new
cases diagnosed. Demographic trends in the next two decades will increase
the population at risk for prostate cancer. Statistics Canada projections
indicate that the population of men over age 50 will increase from 3.9
million in 1999 to 5.6 million in 2011 (44% increase) and 6.3 million in
2016 (62% increase). The United States Census Bureau projections indicate
that the population of men over age 50 will increase, from 33.8 million
in 1999 to 45.8 million in 2011 (36% increase) and 50.7 million in 2016
(50% increase). The American Cancer Society predicts that there will be
about 180,400 new cases of prostate cancer in the United States in the
year 2000, and about 31,900 American men will die of the disease.

[0008]As a consequence of the expected increases in the number of cases of
prostate cancer in the coming years due to rising incidence rates and the
aging North American population, more resources will likely be allocated
to screening men over 50 for this condition, therefore yielding an
increase in the number of cases of identified prostate cancer.

[0009]Prostate cancer often exhibits a long latency period. However, it is
believed that prostate cancer often remains undetected. Also, because it
possesses a high metastatic potential to bone and the lymph nodes, with
<10% of individuals diagnosed with prostate cancer also demonstrated,
by radionuclide scans, to have bone metastasis, prompt detection and
treatment is needed to limit mortality caused by this disease. A recent
review of treatment of prostate cancer is by Pirtskhalaishvilig et al.
(2001, Cancer Practice 9(6):295).

[0010]Increased detection of prostate cancer is due in part to increased
awareness and the widespread use of clinical markers such as prostate
specific antigen (PSA). Prostate specific antigen is a protein that is
produced in very high concentrations in prostate cancer cells. Cancer
development results in an altered and subsequent loss of normal gland
architecture. This in turn leads to an inability to remove secretions and
thus the secretions reach the serum. Serum PSA measurement is one method
for screening for prostate cancer.

[0011]The current diagnostic and treatment paradigm for prostate cancer is
reflected in Clinical Practice Guidelines that are widely available to
practicing physicians. The guidelines presented below outline the common
approach to the detection and management of prostate cancer. [0012]The
Prostate Specific Antigen test is a blood test used to detect prostate
cancer in the earliest stages and should be offered annually to men 50
and older with a life expectancy of 10 years or more, and to younger men
at high risk for prostate cancer. [0013]The Digital Rectal Exam (DRE) is
a test that helps to identify cancer of the prostate, and should be
performed on men who are 50 and older and to younger men at high risk for
prostate cancer. [0014]A biopsy is recommended for all men who have an
abnormal PSA or DRE. [0015]The options for primary management of prostate
cancer are surgery, radiation therapy or close observation. Treatment
decisions are based on the aggressiveness of the cancer, the stage of the
cancer and the life expectancy of the individual. [0016]Advanced prostate
cancer is best managed with hormone therapy. [0017]Radiation therapy can
include external and implanted seeds, a procedure known as brachytherapy.

[0018]The PSA test, which facilitates early detection of prostate cancer,
has been available in Canada since 1986, although its use did not become
widespread until the early 1990's. In 1994 the U.S. Food and Drug
Administration (FDA) approved the use of the PSA test in conjunction with
DRE as an aid in detecting prostate cancer. The free PSA test (fPSA), a
more sensitive test for prostate cancer risk than the standard PSA test,
received FDA approval in 1998.

[0019]Prostate Specific Antigen is an enzyme made by all prostate cells
and normally secreted into semen. Both cancer and a number of benign
conditions can change the architecture of the prostate gland so the
enzyme escapes into the bloodstream. Once there, PSA can exist in two
forms, one that is free-floating and another that is bound to proteins.
The standard PSA test measures both forms. There are a number of
specialized PSA tests which are used to help differentiate between
elevated PSA due to benign conditions and those elevations due to
prostate cancer. The free PSA test evaluates the ratio between the PSA
that is free in the blood and the total PSA (free and protein bound PSA)
in the blood. When the result of the free PSA test is low (i.e. <15%),
there is a higher potential that the individual has prostate cancer. The
PSA velocity is used to describe the speed at which the PSA value
increases over a series of blood tests. The PSA density is used to
evaluate the level of PSA in relation to overall size of the prostate
gland.

[0020]The various PSA tests share some common limitations: [0021]The
principal concern is that although diagnostic accuracy has improved with
each of the modifications to total serum PSA measurement, none of the
forms is specific for prostate cancer. [0022]Each requires a trade-off in
specificity for increased sensitivity and vice versa. This trade-off
appears to be most advantageous with the proportion of free PSA.
[0023]Elevation of PSA may indicate prostate cancer. However, several
other common benign conditions, including Benign Prostatic Hyperplasia
(BPH), are known to be associated with an elevated PSA.

[0024]Because of the limitations of the PSA test (lack of specificity for
prostate cancer and a significant number of "false positive" and "false
negative" test results) it remains an investigational tool as opposed to
an absolute diagnostic test. Abnormal findings following the
administration of the PSA test lead the investigator to perform a biopsy.
Physicians are advised to consider a biopsy to confirm a prostate cancer
diagnosis when a PSA test reading is at least 4.0 ng/mL, when the PSA
level of an individual significantly increases from one test to the next,
or when a DRE is abnormal. A biopsy is recommended for all men who have a
PSA test result above 10 ng/mL.

[0025]The limitations of the PSA test are obvious considering the fact
that only one of four individuals biopsied receives results that are
positive for the presence of cancerous cells. A Canadian study has
estimated the positive predictive value of the PSA test to be as low as
14.4%. This is significant considering the costs associated with a
follow-up biopsy as well as the unnecessary pain and anxiety caused for
individuals.

[0026]Since FDA approval in the U.S., the fPSA test is becoming a
follow-up test for men whose PSA falls in a "diagnostic gray zone" of
moderately elevated levels (4 to 10 ng/mL).

[0027]The digital rectal examination is a simple, inexpensive and direct
method of assessing the prostate, but it is unreliable as a sole
indicator of prostate cancer. The cancer detection rate is higher with
PSA screening than with digital rectal examination (DRE), and the rate
increases when the DRE modality is combined with PSA analysis and/or
transrectal ultrasound examination (TRUS). DRE has never been shown to be
reliable for, staging of prostate cancer. TRUS guided biopsy is required
to follow-up on a positive PSA test in order to help confirm the presence
or absence of disease in the individual's prostate.

[0028]Prostate biopsies are performed to confirm the presence of cancer
cells following suspicion raised by the DRE or a positive PSA test. The
most commonly reported complications of biopsy consist of traces of blood
in the urine, semen or feces. These complications are limited and subside
with 2-3 weeks after the procedure. Pain at the time of biopsy is
universally reported. Only in exceptional cases is analgesia or sedation
required. Most men (>90%) have no significant pain after 24 hours of
the biopsy. Prostate biopsies are costly in the U.S. and may be painful
or psychologically traumatic. Prostatic biopsy represents the cornerstone
of prostate cancer diagnosis.

[0029]For prostate cancers in general, biopsies miss cancers at a rate
estimated as high as 50 percent. Furthermore, even if a cancer is
detected, the location and staging of cancerous cells are not adequately
identified.

[0030]Thus, there is a need for an improved method for diagnosis and/or
detection of cancerous prostate cells.

[0031]An important prognostic factor is prostate cancer stage. Cancer
staging is performed to determine the extent and spread of cancer in the
prostate. Prostate cancer metastasizes by local spread to the pelvic
lymph nodes, seminal vesicles, urinary bladder, or pelvic side walls and
to distant sites such as bone, lung, liver, or adrenals. The
tumor-nodes-metastasis (TMN) staging system is the one most widely used
in North America.

[0032]The limitations of the biopsy in detecting disease and staging a
malignancy is compounded by the fact that prostate cancer is a
heterogeneous disease with apparently independent foci of cancer scatter
throughout the gland. The cancer foci have different malignant potentials
and do not pose equal risks for the individual. Heterogeneity confounds
the interpretation of positive prostate biopsies since it is not possible
to be certain that the most clinically relevant foci of cancer have been
detected.

[0033]Approximately only 30% of early stage disease will progress to
clinically relevant disease within the lifetime of the individual. It is
therefore critical to be able to identify those individuals at risk of
progression who would benefit from aggressive therapy while sparing
low-risk individuals the morbidity resulting from aggressive treatment of
indolent disease. Neither rising PSA nor the presence of cancer cells on
biopsy may indicate definitively the presence of lethal disease.

[0034]Serum PSA is a valuable cancer marker but cannot be used alone to
determine the clinical or pathological stage of prostate cancer or to
identify individuals with potentially curable disease. The combination of
serum PSA with Gleason Score (a grading system for the classification of
adrenocarcinoma of the prostate by observation of the pattern of
glandular differentiation) and clinical stage provides a better
prediction of the final pathologic stage than do any of these variables
separately. Nomograms have been developed and revised to predict the
final pathologic stage based on a combination of serum PSA level, Gleason
Score, and clinical stage. Because these nomograms only offer a
statistical probability of disease organ confinement, further
radiographic evaluation has often been used for the individual. However,
definitive detection of lymph node metastases with standard anatomical
modalities of computed tomography (CT) and magnetic resonance imaging
(MRI) has generally proved ineffective, except for the increasingly more
uncommon cases with large volume soft-tissue involvement (greater than 1
cm) at presentation.

[0035]There is a great need for a new prostate imaging technology that
provides for accurate visualization of extraprostatic growth indicative
of metastasis. Such a technology would provide physicians with a tool to
determine the progression of the cancer and would be extremely valuable
in directing treatment options. Spectroscopy significantly improves the
diagnosis of extracapsular extension by MRI. However, studies demonstrate
that there is high variability in how clinicians interpret the
significance of extracapsular extension. Both CT and MRI can be helpful
in staging prostate cancer, because they can indicate periprostatic
cancer spread, lymph node abnormality and bone involvement, but their
sensitivity for revealing cancer extension has limitations.

[0036]Imaging techniques such as CT or MRI are unable to distinguish
metastatic prostate cancer involvement of lymph nodes by criteria other
than size (i.e. >1 cm). Thus, these imaging techniques, being
inherently insensitive and non-specific, are insufficient for detection
of disease.

[0037]The presence of pelvic lymph node metastasis influences both the
treatment and the prognosis of individuals with prostate cancer. Lymph
node involvement can be assessed surgically. However, incomplete sampling
at the time of radical prostatectomy lead's to false-negative
interpretations in at least 12%, and possibly as many as, 33% of
individuals with lymph node metastases, because isolated metastases in
the external iliac, presciatic, or presacral lymph nodes are outside the
boundaries of the standard Pelvic Lymph Node Dissection.

[0038]Thus, there is a need for a non-invasive test that is able to
identify lymph node metastases in individuals at risk for extraprostatic
disease following the detection of elevated PSA and/or abnormal DRE and a
positive biopsy. This will allow clinicians to reliably differentiate
individuals with organ-confined disease from those with metastatic spread
to lymph nodes. This will provide the opportunity for the individual and
physician to make an informed decision on how to treat the disease and
will significantly improve individual health outcome.

[0039]Despite considerable research into methods for therapy and disease
treatment, prostate cancer remains difficult to treat. Current methods,
commonly based on surgery and/or radiation therapy, are ineffective in a
significant number of cases. Prostate surgery, for example, holds the
potential for damaging nerve tissue and compromising an individual's
chances of recovering sexual function. There is a need for an imaging
technology that can help to minimize the risks involved in surgery by
determining the location of both the cancer and the individual's normal
structures.

[0040]Furthermore, a new technology that is able to localize cancerous
prostate cells that remain following radical prostatectomy would assist
physicians in removing all of the cancerous cells from an individual's
body with focused treatment such as radiation therapy. A labeled
technology that selectively binds prostate cancer cells will allow
clinicians to localize any remaining cancer cells following surgery. An
additional new technology would provide direct delivery of therapeutic
agents, perhaps preventing the need for surgery.

[0041]Thus, there is a need for an improved method to detect and/or
diagnose lymph node metastases in individuals at risk for extraprostatic
disease following the detection of elevated PSA and/or abnormal DRE and a
positive biopsy.

[0042]A substantial amount of work has been put into identifying enzyme or
antigen markers, which could be used as sites for detection and/or
diagnosis for various types of cancers. These markers could also be used
to target cancer cells for treatment with therapeutic and/or cancer cell
killing agents. The ideal cancer marker would exhibit, among other
characteristics, tissue or cell-type specificity.

[0043]A 750 amino acid protein (FIG. 2; SEQ ID NO:22), prostate-specific
membrane antigen (PSMA), localized to the prostatic membrane has been
identified. The complete coding sequence of the gene (FIG. 1; nucleotides
262 to 2514 of GenBank® accession number NM--004476) is presented
as SEQ ID NO:22. PSMA is an integral Type II membrane glycoprotein with a
short intracellular tail and a long extracellular domain. This antigen
was identified as the result of generating monoclonal antibodies to a
prostatic cancer cell, LNCaP (Horoszewicz et al. (1983) Cancer Res.
43:1809-1818). Israeli et al. (Israeli et al. (1993) Cancer Res.
53:227-230) describes the cloning and sequencing of PSMA and reports that
PSMA is prostate-specific and shows increased expression levels in
metastatic sites and in hormone-refractory states. Other studies have
indicated that PSMA is more strongly expressed in prostate cancer cells
relative to cells from the normal prostate or from a prostate with benign
hyperplasia. Current methods of targeting prostate specific membrane
antigen use antibodies with binding specificity to PSMA. One of the first
antibodies described with binding specificity to PSMA was 7E11
(Horoszewicz et al. (1987) Anticancer Res. 7:927-936 and U.S. Pat. No.
5,162,504). Indium-labeled 7E11 localizes to both prostate and sites of
metastasis, and is more sensitive for detecting cancer sites than either
CT or MR imaging, or bone scan (Bander (1994) Sem. In Oncology
21:607-612).

[0044]One of the major disadvantages of the 7E11 antibody is that it is
specific to the portion of the PSMA molecule which is present on the
inside of the cell (intracellular). Antibody molecules do not normally
cross the cell membrane, unless they bind to an extracellular antigen,
which subsequently becomes internalized. As such, 7E11 can not be used to
target a living prostate cell, cancerous or otherwise. The use of 7E11
for detection or imaging is therefore limited to pockets of dead cells
within cancers or tissues with large amounts of dead cells, which cells
render available their intracellular portion of PSMA for binding with
this antibody.

[0045]U.S. Pat. No. 6,107,090, in the name of Neil Bander, and U.S. Pat.
No. 6,150,508, in the name of Gerald Murphy et al. describe numerous
monoclonal antibodies which recognize the extracellular domain of PSMA,
thereby overcoming one of the major drawbacks of the 7E11 antibody. These
antibodies, being able to bind to the extracellular domain of PSMA are
capable of binding to living prostate cells, thereby allowing a more
effective method of diagnosis than 7E11.

[0046]As described above, antibodies to PSMA are already in use for
diagnostic purposes. For example, PSMA is the antigen recognized by the
targeting monoclonal antibody used in ProstaScint®, U.S. Pat. Nos.
5,162,504 and 5,763,202, Cytogen's imaging agent for prostate cancer.

[0047]It would be highly desirable to be provided with an improved
antibody specific for PSMA and a method for diagnosis and/or detection of
cancerous prostate cells.

[0048]It would be highly desirable to be provided with a new prostate
imaging technology offering accurate visualization of extraprostatic
growth indicative of metastasis which would provide physicians with a
tool to determine the progression of the cancer and be extremely valuable
in directing treatment options.

[0049]It would be highly desirable to be provided with a non-invasive test
that is able to identify lymph node metastases in individuals at risk for
extraprostatic disease following the detection of elevated PSA and/or
abnormal DRE and a positive biopsy.

[0050]It would be highly desirable to be provided with an imaging
technology that decreases morbidity by identifying individuals in which
surgery is not indicated.

[0051]It would be highly desirable to be provided with a new technology
that is able to localize cancerous prostate cells that remain following
radical prostatectomy which would assist physicians in removing all of
the cancerous cells from an individual's body. In addition, it would be
highly desirable to be provided with a new technology which would provide
direct delivery of therapeutic agents, perhaps preventing the need for
surgery.

[0052]It would be highly desirable to be provided with an improved method
to detect and/or diagnose lymph node metastases in individuals at risk
for extraprostatic disease following the detection of elevated PSA.

[0053]It would be highly desirable to be provided with a new prostate
imaging technology that provides for accurate visualization of
extraprostatic growth indicative of metastasis which would provide
physicians with a tool to determine the progression of the cancer and be
extremely valuable in directing treatment options.

[0054]It would be highly desirable to be provided with novel antibodies
and their use for detecting, imaging, staging, treating and monitoring of
prostate cancer, and/or metastasis thereof. It would also be highly
desirable to be provided with novel pharmaceutical compositions for the
treatment of prostate cancer.

SUMMARY OF THE INVENTION

[0055]One aim of the present invention is to provide novel antibodies and
their use for detecting, imaging, staging, treating and monitoring of
prostate cancer, and/or metastasis thereof.

[0056]Another aim of the present invention is to provide novel
pharmaceutical compositions for the treatment of prostate cancer.

[0058]Preferably the antigen of the extracellular region of PSMA of the
present invention is from a mammal, more preferably a human.

[0059]In accordance with another embodiment of the present invention there
is provided a peptide selected from the group consisting of SEQ ID
NOs:1-14.

[0060]In accordance with another embodiment of the present invention there
is provided a recombinant nucleic acid molecule comprising a sequence
which encodes a peptide of SEQ ID NOs:1-14, a variant or a fragment
thereof.

[0062]A preferred recombinant DNA molecule of the present invention is
operatively linked to an expression control sequence.

[0063]In accordance with another embodiment of the present invention there
is provided an expression vector containing the recombinant DNA molecule.

[0064]In accordance with another embodiment of the present invention there
is provided a method of expressing a recombinant DNA molecule in a cell
containing the expression vector, comprising culturing the cell in an
appropriate cell culture medium under conditions that provide for
expression of the recombinant DNA molecule by the cell.

[0065]A preferred method of expressing a recombinant DNA molecule in a
cell containing the expression vector further comprises the step of
purifying a recombinant product of the expression of the recombinant DNA
molecule.

[0066]In accordance with another embodiment of the present invention there
is provided a unicellular host transformed with a recombinant DNA
molecule for expression of a peptide of SEQ ID NOs:1-14, a variant or a
fragment thereof.

[0067]In accordance another embodiment of with the present invention there
is provided an immunogenic composition for raising antibodies specific to
PSMA in a subject, which comprises a peptide selected from the group
consisting of SEQ ID NOs:1-14 modified with an immunogenic moiety or
carrier and/or an antigen of the present invention in association with a
pharmaceutically acceptable carrier.

[0068]In a preferred immunogenic composition of the present invention the
subject is an animal selected from the group consisting of mammals and
birds, more preferably a human or a mouse, such as a BALB/c mouse, or a
rabbit.

[0069]In a preferred immunogenic composition the immunogenic moiety or
carrier is selected from the group consisting of keyhole limpet
hemocyanin (KLH) and bovine serum albumin (BSA).

[0070]In accordance with another embodiment of the present invention there
is provided a method of raising antibodies which bind to PSMA, which
comprises administering an immunogenic amount of an immunogenic
composition of the present invention, such as PSMA, an epitope of PSMA,
or intact cell and/or fragment thereof exhibiting the extracellular
region of PSMA, to an animal.

[0071]In accordance with another embodiment of the present invention there
is provided a method of producing antibodies which bind to PSMA,
comprising treating an animal with an immunogenic amount of an
immunogenic composition of the present invention, such as PSMA, an
epitope of PSMA, or intact cell and/or fragment thereof exhibiting the
extracellular region of PSMA, to produce antibodies; and isolating the
antibodies from serum of the animal.

[0072]In accordance with another embodiment of the present invention there
is provided an isolated antibody or antigen binding fragment thereof,
which binds to an antigen of the present invention.

[0073]A preferred isolated antibody or antigen binding fragment thereof of
the present invention is a monoclonal antibody, such as a monoclonal
antibody selected from the group consisting of F34-8H12, F42-3E11,
F42-17G1, F42-29B4, F42-30C1 AND F47-20F2, or a polyclonal antibody.

[0074]The binding fragment may be selected from the group consisting of a
Fab fragment, a F(ab')2 fragment, and a Fv fragment.

[0075]In accordance with another embodiment of the present invention there
is provided a pharmaceutical composition for targeted treatment of
prostate cancer, and/or metastasis with PSMA thereon, which comprises an
antibody or binding fragment thereof according to the present invention
bound to a cytotoxic drug in association with a pharmaceutically
acceptable carrier, wherein the PSMA binding site of the antibody is
available for targeted binding to PSMA and the bound cytotoxic drug
remains biologically active.

[0076]In a preferred pharmaceutical composition of the present invention
the cytotoxic drug is selected from the group consisting of iodine-125,
iodine-131, cyclophosphamide, taxol, IFN-alpha and IL2 and/or mixtures
thereof.

[0077]In accordance with another embodiment of the present invention there
is provided a method for treating prostate cancer, and/or metastasis
thereof comprising administering to an individual a pharmaceutically
effective amount of a pharmaceutical composition according to the present
invention.

[0078]In a preferred method of the present invention the administering is
carried out orally, rectally, parenterally, subcutaneously,
intravenously, intramuscularly, intraperitoneally, intraarterially,
transdermally or by application to a mucus membrane.

[0079]In accordance with another embodiment of the present invention there
is provided a composition for detection of prostate cancer, and/or
metastasis thereof with PSMA thereon in an individual and/or in a sample
obtained therefrom, which comprises an antibody or binding fragment
thereof according to the present invention bound to a detectable label in
association with a physiologically acceptable carrier or an in vitro
acceptable carrier, wherein the PSMA binding site of the antibody is
available for binding to PSMA and the detectable label remains
detectable.

[0080]In a preferred composition of the present invention the detectable
label is selected from the group consisting of a radioactive label, a
fluorescent label, a nuclear magnetic resonance active label, a
luminescent label, a chromophore label, a positron emitting isotope for
PET scanner, chemiluminescence label, or an enzymatic label.

[0081]In accordance with another embodiment of the present invention there
is provided a method of detecting prostate cancer cell, and/or metastasis
thereof in an individual comprising administering to the individual an
effective amount of a composition according to the present invention or
subjecting a biological sample obtained from the individual to an
effective amount of the composition according to the present invention
and detecting the signal produced by the detectable label, wherein
detection of the label above a certain level is indicative of the
presence of prostate cancer, and/or metastasis thereof. A preferred
method of the embodiment of present invention further comprises
localizing a detectable label within the individual or a sample obtained
therefrom.

[0082]In a preferred method of the present invention a 2-dimensional
and/or 3-dimensional image of the individual or a sample obtained
therefrom is generated.

[0083]In a preferred method of the present invention the method is used to
indicate the location of prostate cancer, and/or metastasis thereof
within the individual and/or sample obtained therefrom.

[0084]In accordance with another embodiment of the present invention there
is provided an assay system for detecting prostate cancer, and/or
metastasis thereof comprising a labeled antibody and/or antigen binding
fragment thereof according to the present invention.

[0085]A preferred assay of the present invention further comprises means
for semi-quantifying or quantifying an amount of antigen bound to the
antibody and/or antigen binding fragment thereof, wherein an amount of
antigen bound to the antibody and/or antigen binding fragment thereof
above a predetermined level is indicative of prostate cancer, and/or
metastasis thereof.

[0086]In a preferred assay of the present invention the assay is selected
from the group consisting of immunoassay, enzyme linked immunosorbent
assay (ELISA), array-based immunoassay, array-based ELISA.

[0087]A preferred assay of the present invention further comprises means
for receiving the biological sample.

[0088]A preferred assay of the present invention further comprises a
multi-well microplate including the antibody and/or antigen binding
fragment thereof in at least one well.

[0089]In a preferred assay of the present invention the antibody and/or
antigen binding fragment thereof binds to a peptide selected from the
group consisting of PSMA, an extracellular region of PSMA, a peptide
corresponding to an extracellular region of PSMA, an epitope of PSMA, and
SEQ ID NOs:1-14.

[0090]In accordance with another embodiment of the present invention there
is provided a method of determining relative efficacy of a therapeutic
regimen to be performed on an individual suffering from and/or being
treated for prostate cancer, and/or metastasis thereof, the method
comprising: (a) initially analyzing the individual or a biological sample
obtained therefrom to determine presence of cancer-associated antigen
able to bind with the antibody and/or antigen binding fragment thereof
according to the present invention; and (b) periodically repeating step
(a) during treatment of the individual to determine an increase or
decrease in quantity of cancer-associated antigen present in the sample.

[0091]In accordance with another embodiment of the present invention there
is provided a method of determining the recurrence of a prostate cancer
disease state in an individual clinically diagnosed as stabilized or in a
remissive state, the method comprising analyzing the individual or a
biological sample obtained therefrom to quantitate cancer-associated
antigen immunoreactive with an antibody and/or antigen binding fragment
thereof according to the present invention.

[0092]In accordance with another embodiment of the present invention there
is provided a kit for detecting prostate cancer, and/or metastasis
thereof comprising a composition according to the present invention.

[0094]For the purpose of the present invention the following terms are
defined below.

[0095]The term "cancer" is intended to mean any cellular malignancy whose
unique trait is the loss of normal controls which results in unregulated
growth, lack of differentiation and ability to invade local tissues and
metastasize. Cancer can develop in any tissue of any organ. More
specifically, cancer is intended to include, without limitation, prostate
cancer, leukemia, hormone dependent cancers, breast cancer, colon cancer,
lung cancer, epidermal cancer, liver cancer, esophageal cancer, stomach
cancer.

[0096]The term "prostate cancer" is intended to mean an uncontrolled
(malignant) growth of cells in the prostate gland, which is located at
the base of the urinary bladder and is responsible for helping control
urination as well as forming part of the semen.

[0097]The term "metastasis" is intended to mean cancer that has spread
beyond the prostate. "Metastasis" is also intended to mean the process by
which cancer spreads from one part of the body to another, the way it
travels from the place at which it first arose as a primary tumor to
distant locations in the body.

[0098]The term "antibody" (Ab) is intended to mean intact antibody
molecules as well as fragments, or binding regions or domains thereof
(such as, for example, Fab, F(ab')2 and Fv fragments) which are
capable of binding an antigen. Such fragments are typically produced by
proteolytic cleavage, with enzymes such as papain or pepsin.
Alternatively, antigen-binding fragments can be produced through
recombinant DNA technology or through synthetic procedures.

[0099]The term "monoclonal antibody" (mAb) is intended to mean an antibody
produced by a single clone of cells or a cell line derived from a single
cell that has unique antigen binding characteristics or recognizes an
individual molecular target. Such antibodies are all identical and have
unique amino acid sequences.

[0100]The term "epitope" is intended to mean a molecular region on the
surface of an antigen capable of eliciting an immune response and of
combining with the specific antibody produced by such a response.

[0101]The term "cytotoxic compound" is intended to mean a compound, or
molecule which is capable of killing a cell.

[0102]The term "detectable label" is intended to mean a label effective at
permitting detection of a cell or portion thereof upon binding of a
molecule to which the detectable label is attached to said cell or
portion thereof. Alternatively, the detectable label permits detection of
a cell upon internalization of the detectable label by the cell. A
detectable label includes but is not limited to a radioactive label, a
fluorescent label, a nuclear magnetic resonance active label, a
luminescent label, a chromophore label, a positron emitting isotope for
PET scanner, chemiluminescence label, or an enzymatic label.

[0103]The term "biological sample" is intended to mean a sample obtained
from an individual and includes, but is not to be limited to, any one of
the following: tissue, cerebrospinal fluid, plasma, serum, saliva, blood,
nasal mucosa, urine, synovial fluid, microcapillary microdialysis.

[0104]The terms "treatment", "treating" and the like are intended to mean
obtaining a desired pharmacologic and/or physiologic effect, such as
inhibition of cancer cell growth or induction of apoptosis of a cancer
cell. The effect may be prophylactic in terms of completely or partially
preventing a disease or symptom thereof and/or may be therapeutic in
terms of a partial or complete cure for a disease and/or adverse effect
attributable to the disease. "Treatment" as used herein covers any
treatment of a disease in a mammal, particularly a human, and includes:
(a) preventing a disease or condition (e.g., preventing cancer) from
occurring in an individual who may be predisposed to the disease but has
not yet been diagnosed as having it; (b) inhibiting the disease, (e.g.,
arresting its development); or (c) relieving the disease (e.g., reducing
symptoms associated with the disease).

[0105]The terms "administering" and "administration" are intended to mean
a mode of delivery including, without limitation, oral, rectal,
parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal,
intraarterial, transdermally or via a mucus membrane. The preferred one
being orally. One skilled in the art recognizes that suitable forms of
oral formulation include, but are not limited to, a tablet, a pill, a
capsule, a lozenge, a powder, a sustained release tablet, a liquid, a
liquid suspension, a gel, a syrup, a slurry, a suspension, and the like.
For example, a daily dosage can be divided into one, two or more doses in
a suitable form to be administered at one, two or more times throughout a
time period.

[0106]The term "therapeutically effective" is intended to mean an amount
of a compound sufficient to substantially improve some symptom associated
with a disease or a medical condition. For example, in the treatment of
cancer, a compound which decreases, prevents, delays, suppresses, or
arrests any symptom of the disease would be therapeutically effective. A
therapeutically effective amount of a compound is not required to cure a
disease but will provide a treatment for a disease such that the onset of
the disease is delayed, hindered, or prevented, or the disease symptoms
are ameliorated, or the term of the disease is changed or, for example,
is less severe or recovery is accelerated in an individual.

[0107]The compounds of the present invention may be used in combination
with either conventional methods of treatment and/or therapy or may be
used separately from conventional methods of treatment and/or therapy.

[0108]When the compounds of this invention are administered in combination
therapies with other agents, they may be administered sequentially or
concurrently to an individual. Alternatively, pharmaceutical compositions
according to the present invention may be comprised of a combination of a
compound of the present invention, as described herein, and another
therapeutic or prophylactic agent known in the art.

[0109]It will be understood that a specific "effective amount" for any
particular individual will depend upon a variety of factors including the
activity of the specific compound employed, the age, body weight, general
health, sex, and/or diet of the individual, time of administration, route
of administration, rate of excretion, drug combination and the severity
of the particular disease undergoing prevention or therapy.

[0110]As used herein, "pharmaceutically acceptable carrier" includes any
and all solvents (such as phosphate buffered saline buffers, water,
saline), dispersion media, coatings, antibacterial and antifungal agents,
isotonic and absorption delaying agents and the like. The use of such
media and agents for pharmaceutically active substances is well known in
the art. Except insofar as any conventional media or agent is
incompatible with the active ingredient, its use in therapeutic
compositions is contemplated. Supplementary active ingredients can also
be incorporated into the compositions.

[0119]Some epitopes were chosen based on hydrophilic character of the
amino acid sequence (SEQ ID NO:22) and the lack of glycosylation
consensus sites. Other sequences were selected from a rigorous analysis
of PSMA secondary structure prediction and homology modeling with the
most similar protein crystal structure (human transferrin receptor type
1). Regions were selected according to their apparent high solvent
accessibility, flexibility, and coiled coil structure. In all cases the
aim was to optimize antigenicity and sequence uniqueness such that
antibodies raised against these peptides do not likely cross-react with
other proteins.

[0120]In accordance with the present invention, there is provided a
peptide corresponding to an epitope of the extracellular region of PSMA
selected from the group consisting of SEQ ID NOs:1-14.

[0121]Small molecules such as the peptides of the present invention are
incomplete immunogens. Although they are able to react specifically with
antibodies, they are unlikely to induce an immune response when they are
injected into an animal. In order to make them immunogenic in animals,
small peptide sequences are covalently coupled to a carrier molecule,
such as keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA).
KLH and BSA are coupled to the peptides via a cysteine amino acid residue
added to the N-terminus of the sequence of each peptide. The resulting
peptide-conjugates are used to raise polyclonal and monoclonal
antibodies.

[0122]In accordance with the present invention, there is provided an
immunogenic peptide or recombinant peptide or protein for raising
antibodies specific to PSMA, which comprises a peptide corresponding to
an epitope of the extracellular region of PSMA modified with an
immunogenic moiety or carrier.

[0123]In accordance with the present invention, there is provided a method
for raising antibodies which bind to the epitopes and peptides of the
present invention, which also have binding specificity to PSMA, such as
PSMA in its native environment in LNCaP cells, or recombinant PSMA. The
antibodies, or binding portions thereof, recognize and bind to PSMA in
normal, benign, hyperplastic and cancerous prostate cells. Moreover, the
antibodies, or binding portions thereof recognize and bind to PSMA in
living normal, benign, hyperplastic and cancerous prostate cells. As a
result of this binding, the antibodies or binding portions thereof are
concentrated in areas with large numbers of prostate cells or portions
thereof.

[0124]Antibodies in accordance with the present invention may be produced
by procedures generally known in the art. For example, polyclonal
antibodies may be produced by injecting the peptide or protein, such as
PSMA or purified recombinant PSMA, alone or coupled to a suitable
immunogenic moiety or carrier into a non-human animal. After an
appropriate period, the animal is bled, sera recovered and purified by
techniques known in the art. Monoclonal antibodies may be prepared, for
example, by the Kohler-Milstein technique (1975, Nature
256(5517):497-497) involving fusion of an immune B-lymphocyte to myeloma
cells. For example, antigen as described above can be injected into mice
as described above until a polyclonal antibody response is detected in
the mouse's sera. The mouse can be boosted again, its spleen removed and
fusion with myeloma conducted according to a variety of methods. The
individual surviving hybridoma cells are tested for the secretion of
antibodies which bind the extracellular region of PSMA first by their
ability to bind the immunizing antigen (peptide/protein). Monoclonal
antibodies are produced in large quantities by growing the hybridoma
clones in vitro or in vivo.

[0125]Serum from immunized and nonimmunized (control) animals are tested
for the presence of specific antibodies in an Enzyme Linked ImmunoSorbent
Assay (ELISA). For the ELISA assay each peptide is covalently coupled to
a carrier molecule different than that used in the immunization phase of
the procedure, or used uncoupled. Such a carrier molecule is, for
example, bovine serum albumin (BSA). The same N-terminal cysteine of each
peptide used to couple to the carrier molecule used for raising
antibodies, for example KLH, is used to couple to the carrier molecule
used for the ELISA, for example BSA. There are two reasons for this.
First, immunization of animals with peptide-KLH induces the production of
antibodies to both the peptide and KLH. Therefore, when screening for
antibodies to the peptide it is important to eliminate the possibility of
detecting binding to the KLH carrier by using peptide linked to a carrier
the immunized mice have never seen. This eliminates background reactivity
in the assay that may mask reactivity to the peptide of interest. Second,
linking peptide to BSA in a similar manner as it was linked to KLH should
permit antibodies induced to the peptide by immunization with peptide-KLH
to recognize that peptide linked to the BSA carrier because its
orientation is the same on each carrier surface.

[0126]The processes of the present invention encompass both whole
antibodies and the binding portions thereof. Such binding portions
thereof include Fab fragments, F(ab')2 fragments, and Fv fragments.
These antibody fragments can be prepared by conventional procedures, such
as proteolytic fragmentation as described in J. Goding, Monoclonal
Antibodies: Principles and Practice, pp. 98-118, N.Y. Academic Press
1983.

[0127]Preferred monoclonal antibodies in accordance with one embodiment of
the present invention are identified in Table 1 below. These antibodies
were raised using peptide PS0215 (SEQ ID NO:8).

[0128]The antibody or binding portion thereof of the present invention can
be used alone or in combination as a mixture with at least one other
antibody or binding portion thereof with binding specificity for prostate
antigen not herein described.

[0130]In accordance with the present invention, there is provided a
monoclonal antibody or binding fragment thereof which binds to a peptide
corresponding to an epitope of the extracellular region of PSMA selected
from the group consisting of SEQ ID NOs:1-14.

[0132]In accordance with the present invention there, is provided a
hybridoma cell line that produces a monoclonal antibody which binds to a
peptide corresponding to an epitope of the extracellular region of PSMA
selected from the group consisting of SEQ ID NOs:1-14.

[0133]The antibody or binding fragment thereof, or mixtures thereof may be
unmodified or may be linked to 1) a radioimaging agent, such as those
emitting radiation, for detection of the prostate cancer, and/or
metastasis thereof upon binding of the antibody or binding fragment
thereof, or mixtures thereof to the antigen, or 2) a cytotoxic agent,
which kills the prostate cancer, and/or metastasis thereof upon binding
of the antibody or binding fragment thereof, or mixtures thereof to the
antigen. Alternatively, the cytotoxic agent is not toxic until
internalized by the cell. Alternatively, the cytotoxic agent is toxic
whether internalized or not internalized. Treatment is effected by
administering the antibody or binding fragment thereof, or mixtures
thereof to the individual under conditions which allow binding of the
antibody or binding fragment thereof, or mixtures thereof to the antigen,
and which binding results in the death of the cell of the prostate
cancer, and/or metastasis thereof. In a preferred embodiment,
administration is carried out on a living mammal. Such administration can
be carried out orally or parenterally. In another embodiment the method
is used to prevent or delay development or progression of prostate
cancer, and/or metastasis thereof.

[0134]A cytotoxic agent of the present invention can be an agent emitting
radiation, a cellular toxin (chemotherapeutic agent) and/or biologically
active fragment thereof, and/or mixtures thereof to allow cell killing. A
cytotoxic agent such as a cellular toxin and/or biologically active
fragment thereof can be a synthetic product or a product of fungal,
bacterial or other microorganism, such as mycoplasma, viral etc., animal,
such as reptile, or plant origin. A cellular toxin and/or biologically
active fragment thereof can be an enzymatically active toxin and/or
fragment thereof, or can act by inhibiting or blocking an important
and/or essential cellular pathway or by competing with an important
and/or essential naturally occurring cellular component.

[0135]Cytotoxic agents emitting radiation for use in the present invention
are exemplified by Yttrium-90 (Y90), iodine-125 (I125),
iodine-131 (I131) and gamma-emitting isotopes used, for example, to
destroy thyroid tissue in some individuals suffering from
hyperthyroidism.

[0136]Radioimaging agents emitting radiation (detectable radio-labels) for
use in the present invention are exemplified by indium-111 (In111),
technitium-99 (Tc99), or iodine-131 (I131).

[0137]Detectable labels (non-radioactive labels) for use in the present
invention can be a radioactive label, a fluorescent label, a nuclear
magnetic resonance active label, a luminescent label, a chromophore
label, a positron emitting isotope for PET scanner, chemiluminescence
label, or an enzymatic label. Fluorescent labels are exemplified by
fluorescein, and rhodamine. Chemiluminescence labels are exemplified by
luciferase. Enzymatic labels are exemplified by peroxidase and
phosphatase.

[0139]In accordance with the present invention there is provided a
pharmaceutical composition for targeted treatment of prostate cancer,
and/or metastasis with PSMA thereon, which comprises an antibody or
binding fragment thereof, or mixtures thereof bound to a cytotoxic agent
in association with a pharmaceutically acceptable carrier, wherein the
PSMA binding site of the antibody is available for targeted binding of
PSMA and the cytotoxic agent remains biologically active.

[0140]In accordance with the present invention, there is provided a method
of detecting normal, benign, hyperplastic and cancerous prostate
epithelial cells, and/or metastases thereof in an individual or a
biological sample obtained therefrom, i.e., the detection may be in vivo
or in vitro. The method: involves providing an antibody or binding
fragment thereof or or mixtures thereof with binding specificity to an
antigen of prostate cancer, or metastasis thereof. The antibody or
binding fragment thereof or mixtures thereof is bound to a detectable
label which upon binding of the antibody or binding fragment thereof or
mixtures thereof allows detection of the prostate cancer, and/or
metastasis thereof. Detection is effected by administering the antibody
or binding fragment thereof or mixtures thereof to the individual or by
contacting a biological sample obtained therefrom under conditions which
allow binding of the antibody or binding fragment thereof or mixtures
thereof to the antigen. Prostate cancer, and/or metastasis thereof is
detected by monitoring of the signal produced by the detectable label
above a predetermined base level, which indicates the presence of
prostate cancer, and/or metastasis thereof. In a preferred embodiment,
administration is carried out on a living mammal.

[0141]Detection of PSMA in, for example, a fluid sample obtained from an
individual is an indication that prostate cells are being lyzed. Since
PSMA is not present in the extracellular fluid of healthy individuals,
the detection of PSMA in a biological sample from an individual is an
indication of prostate cell lysis.

[0142]In a preferred embodiment detection of the signal produced by the
detectable label is used in the generation of a 2-dimensional and/or
3-dimensional image of the individual or a biological sample obtained
therefrom. In another preferred embodiment the 2-dimensional and/or
3-dimensional image is used to indicate the location of prostate cancer,
and/or metastasis thereof within the individual or a biological sample
obtained therefrom.

[0143]In accordance with the present invention there is provided a
composition for targeted detection of prostate cancer, and/or metastasis
thereof with PSMA thereon, which comprises an antibody or binding
fragment thereof or mixtures thereof bound to a detectable label in
association with a physiologically acceptable carrier, wherein said PSMA
binding site of said antibody is available for targeted binding of PSMA
and said detectable label remains detectable from inside or outside a
cell.

[0144]In accordance with the present invention there is provided a method
of detecting prostate cancer, and/or metastasis thereof in an individual
or a biological sample obtained therefrom comprising: administering to
the individual or a biological sample obtained therefrom an effective
amount of a composition which comprises an antibody or binding fragment
thereof or mixtures thereof bound to a detectable label in association
with a physiologically acceptable carrier, wherein the PSMA binding site
of the antibody is available for targeted binding of PSMA and the
detectable label remains detectable from inside or outside a cell; and
detecting the signal produced by the detectable label, wherein detection
of the label above a certain level indicates the presence of prostate
cancer, and/or metastasis thereof.

[0145]The antibody or binding fragment thereof or mixtures thereof with
binding specificity to an antigen of prostate cancer, and/or metastases
thereof of the present invention can be used and sold together with
equipment, as a kit, to detect the particular label.

[0146]In accordance with the present invention there is provided an assay
system for detecting prostate cancer, and/or metastasis thereof
comprising: means for receiving a biological sample; means for detecting
presence of antigen bound to at least one antibody or binding fragment
thereof or mixtures thereof; and means for quantifying an amount of
antigen bound to said at least one antibody or binding fragment thereof
or mixtures thereof, wherein an amount of antigen bound to said at least
one antibody or binding fragment thereof or mixtures thereof above a
predetermined level indicates prostate cancer, and/or metastasis thereof.

[0147]In accordance with the present invention there is provided a method
of determining the relative efficacy of a therapeutic regimen performed
on an individual treated for prostate cancer, and/or metastasis thereof,
the method comprising: initially analyzing an individual or a biological
sample obtained therefrom to quantitate cancer-associated antigen able to
bind with at least one antibody or binding fragment thereof or mixtures
thereof; and periodically repeating the previous step during the course
of application of the therapeutic regimen to determine increase or
decrease in quantity of cancer-associated antigen present in the sample.

[0148]In accordance with the present invention there is provided a method
of determining the recurrence of a prostate cancer disease state in an
individual clinically diagnosed as stabilized or in a remissive state,
the method comprising: initially analyzing an individual or a biological
sample obtained therefrom to quantitate cancer-associated antigen
immunoreactive with at least one antibody or binding fragment thereof or
mixtures thereof; and periodically repeating the previous step during the
course of application of the therapeutic regimen to determine increase or
decrease in quantity of cancer-associated antigen present in the sample.

[0149]Regardless of whether the antibody or binding fragment thereof, or
mixtures thereof of the present invention is used for treatment,
detection, or imaging, it can be administered orally, parenterally,
subcutaneously, intravenously, intramuscularly, intraperitoneally, by
intranasal instillation, by intracavitary or intravesical instillation,
intraocularly, intraarterially, intralesionally, as an aerosol, or by
application to mucous membranes, such as, that of the nose, throat, and
bronchial tubes. It may be administered alone on with a pharmaceutically
or physiologically acceptable carrier, excipient, or stabilizer, and can
be in solid or liquid form such as, tablet, capsule, powder, solution,
suspension or emulsion.

[0150]The treatment and/or therapeutic use of the antibody of the present
invention can be used in conjunction with other treatment and/or
therapeutic methods. Such other treatment and/or therapeutic methods
include surgery, radiation, cryosurgery, thermotherapy, hormone
treatment, chemotherapy, vaccines, other immunotherapies, and other
treatment and/or therapeutic methods which are regularly described.

[0151]In addition to methods of treatment and/or therapeutic use, the
antibodies of the present invention, by their binding positions on the
PSMA protein, can be used for epitope mapping of the architecture of the
PSMA protein in epitope mapping studies. The antibodies of the present
invention can also be used as probes for screening a library of
molecules, agents, proteins, peptides and/or chemicals to identify a
molecule, agent, protein, peptide and/or chemical. Such a library could
be a chemical library, antibody library, phage display library, peptide
library or library of natural compounds. The identified molecule, agent,
protein, peptide and/or chemical could be an antagonist or agonist of
PSMA.

[0152]The present invention will be more readily understood by referring
to the following examples which are given to illustrate the invention
rather than to limit its scope.

Example 1

Peptide Synthesis

[0153]Example 1 relates to the procedures whereby peptides corresponding
to epitopes of the extracellular domain of PSMA are synthesized.

[0154]Table 2 shows the sequence and their location within the PSMA amino
acid sequence of the 14 peptides that were synthesized by solid phase
F-MOC chemistry to greater than 85% purity. Each peptide was synthesized
with a single amino terminal unblocked cysteine residue. This amino acid
was used to conjugate each peptide to lysine residues in KLH and bovine
serum albumin (BSA) carrier proteins using N-maleimide chemistry.

[0155]Example 2 relates to preparation of mouse monoclonal antibodies with
specificity to the peptides of Example 1.

[0156]Several strategies were used to immunize BALB/c mice for production
of PSMA-specific antibodies.

[0157]One strategy consisted of priming and boosting at 2 to 3 week
intervals with peptide conjugated to KLH by one of 2 methods that link
the amino terminal cysteine of the peptide immunogen to lysine residues
on KLH. Peptides were conjugated to KLH using either sulfo-GMBS or SMCC
conjugation systems. This strategy was designed to induce and amplify
peptide specific antibodies.

[0158]A second strategy employed 2 immunizations at 2 to 3 week intervals
with LNCaP membrane followed by 3 immunizations with purified PSMA or
peptide conjugated KLH. Priming with LNCaP membrane should induce the
production of an antibody response directed to membrane antigens
including PSMA presented in a native conformation within a cellular
membrane. Boosting with purified PSMA antigen should further activate and
expand the B lymphocyte clones secreting antibody that recognizes
epitopes present on whole native PSMA whereas boosting with peptide
conjugated KLH should further activate and expand the B lymphocyte clones
recognizing the epitopes corresponding to the peptide used in the boost
immunizations.

[0159]All immunizations were intraperitoneal injections of 100 μl
volumes containing 25 to 50 μg of peptide antigen or 50 μl of LNCap
membrane preparation. The antigen for the first immunization was
emulsified in complete Freund's adjuvant (CFA). Antigen used for
subsequent immunizations was emulsified in incomplete Freund's adjuvant
(IFA). The final boost before fusing donor spleen with the NSO myeloma
parental cell line was done 3 to 5 days before fusion. For this
immunization antigen was diluted in phosphate buffered saline (PBS).

[0160]The fusion was performed according to the technique known in the art
(Kohler G. and Milstein C. (1975) Nature 256 (5517):495-97).

[0161]Supernatants of the resulting wells exhibiting growth were screened
by Enzyme Linked Immunosorbent Assay (ELISA) for the presence of
antibodies binding to peptide (conjugated or not to BSA) and either LNCaP
cell membranes or recombinant PSMA. Negative controls for the screening
step were BSA alone (control for peptide or PSMA binding) or PC-3 cell
membrane (control for LNCaP binding). Wells containing antibodies with
desirable binding characteristics were subjected to at least 2 cycles of
cloning by limiting dilution. Hybridomas secreting either one of the 6
monoclonal antibodies against peptide PS0215 (SEQ ID NO:8) were generated
according to this screening strategy. The isotype of the immunoglobulin
secreted into cultured supernatants by cloned antibody secreting
hybridomas was determined using Isostrips (Roche Diagnostics Corp.,
Indianapolis Ind.).

Example 3

Preparation of Cell Membrane and Purified PSMA

Cell Membrane Preparation

[0162]Example 3 relates to the purification of recombinant PSMA and cell
membrane for immunization and characterization of mAb.

[0164]Total RNA from LNCaP was isolated using the Trizol method according
to manufacturer's directions (GIBCO Life Technologies Inc.) and treated
with DNase I (RNase free). LNCaP RNA was reverse transcribed by
Thermoscript reverse transcriptase and oligo dT primers (GIBCO Life
Technologies Inc.). DNA corresponding to the gene encoding PSMA was then
amplified by PCR using the oligonucleotides (5'3')
ATGTGGAATCTCCTTCACGAAACC (SEQ ID NO:15) and TTAGGCTACTTCACTCAAAGTCTC (SEQ
ID NO:16). The resulting PCR product was cloned into plasmid pCRT7-NT.
Clones were sequenced to verify the identity of the insert DNA as
originating from PSMA.

Baculovirus Expression of PSMA

[0165]PSMA was PCR-amplified from a sequence-confirmed recombinant plasmid
of pCRT7-NT using primers GGGGATCCATGTGGAATCTCCTTCACG (SEQ ID NO:17) and
GGGCTCGAGGGCTACTTCACTCAAAGTCT (SEQ ID NO:18) (full length PSMA, fIPSMA)
or the oligonucleotides GGGGATCCGAAATCCTCCAATGAAGCTACTAAC (SEQ ID NO:19)
and GGGCTCGAGTTAGGCTACTTCACTCAAAGTCTC (SEQ ID NO: 20) (soluble PSMA,
sPSMA). The PCR fragment was digested overnight with the restriction
enzymes BamHI and XhoI and cloned into Novagen transfer vector pBAC-1
(fIPSMA) or pBAC-3 (sPSMA). The recombinant virus encoded either a full
length PSMA containing a C-terminal poly-histidine tag or a truncated
PSMA containing a poly-histidine tag at the N-terminus. Sf9 cells were
co-transfected with the transfer vector DNA and the linearized viral DNA
according to the manufacturer's directions. The viruses were plaque
purified prior amplification to obtain a high titer viral stock.

[0166]Sf9 cells were propagated in TNM-FH medium supplemented with 10%
fetal bovine serum, 0.1% Pluronic F-68 (InVitrogen), and the antibiotics
kanamycin (30 ug/ml), neomycin (20 ug/ml) and streptomycin (200 ug/ml).
Infection of Sf9 cells with recombinant baculovirus was done at a
multiplicity of infection of about 10. After 3 days post-infection.
flPSMA was solubilized from a cell lysate (PBS containing 1% Triton
X-100) and secreted sPSMA was recovered directly from the medium. Both
proteins were purified by affinity chromatography using a Ni-NTA resin,
according to the manufacturer's instruction (Qiagen). The eluate was
dialysed extensively against PBS before use as an immunogen or for
hybridoma screening.

Example 4

Characterization of Monoclonal Antibodies

Monoclonal Antibodies Reactivity to PSMA by ELISA

[0167]Example 4 relates to the characterization of the mAbs by ELISA,
western blot IHC, and in vivo biodistribution.

[0168]mAb reactivity to PSMA was assayed by ELISA. The LNCaP cell line was
used as a source of natural PSMA and various PSMA non-expressing cell
line as negative control. 5 ug of cell membrane preparation in 100 ul PBS
were adsorbed onto 96 well plates (Immulon 2HB, Thermo Labs System)
overnight at 4° C., or 2 hours at room temperature. The plates
were washed with TBST (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05%
Tween-20) then incubated with TBST containing 3% casein for 1 hours to
block non-specific sites. The wells were loaded with 100 ul of the
hybridoma cell supernatants or a dilution in TBST, and incubated for 1
hour at room temperature under gentle agitation. In some cases, the mAb
was pre-mixed with dilutions of the antigenic peptide or an irrelevant
peptide and then the solution applied to coated cell lysate. The plates
were washed with TBST then incubated for 1 hour with a horse-radish
peroxidase conjugated goat anti-mouse IgG (Jackson #115-035-164)
secondary antibody at a dilution 1/1000 in TBST. After extensive washing,
the plates were incubated with 100 ul of the peroxidase substrate TMB
(BioFX). The reaction was stopped with an equivalent volume of 0.5N
sulfuric acid and the reactivity evaluated by reading at OD 450 nm.

[0169]FIG. 3 shows a representative reactivity of the six monoclonal
antibodies for the LNCaP cells (-quadrature-) compared to the PSMA
negative human cancer cell lines PC-3 (prostate, -Δ-), K562
(myeloid leukemia, -x-) and NMB-7-(neuroblastoma, -Δ-). The graph
illustrates that only a very weak signal was detected from the negative
control cell lines as compared to the strongly reactive LNCaP cells.
Indeed, the average reactivity (±SEM) of the antibodies to LNCaP over
PC-3 background was found to be 9.0±3.6 for the 8H12 (n=8),
25.7±6.3 for the 3E11 (n=7), 26.1±6.32 for the 29B4 (n=8),
10.9±3.0 for the 30C1 (n=5), 16.9±4.4 for the 17G1 (n=5), and
58.9±15.6 for the 20F2 (n=4). These results suggest that the
reactivity of the mAbs is specific for a protein expressed by the LNCaP
cells only.

[0170]In order to confirm the specificity of the mAbs, the reactivity of
the mAbs to LNCaP cells were challenged by the original antigen from
which they were generated (PS0215) (SEQ ID NO:8). FIG. 4 shows that
nanomolar concentrations of the antigenic peptide PS0215 (-quadrature-)
can completely inhibits the binding of the antibodies to LNCaP cells. In
contrast, no change in the reactivity of the antibodies were observed
when challenged with up to micromolar concentration of another peptide
derived from the PSMA amino acid sequence (PS0210, -∘-). The
results suggests that the antibodies recognize a unique linear amino acid
sequence of PSMA (PS0215) i.e. corresponding to PS0215 or SEQ ID NO:8.

Western Blot Detection of PSMA

[0171]Western Blot analysis were performed on LNCaP and PC-3 cell membrane
in order to confirm that the mAbs detect the PSMA protein. Proteins from
2.5 ug of a cell membrane preparation were separated by
SDS-polyacrylamide gel electrophoreisis on a 7.5% gel. The proteins were
then transferred to a PVDF membrane and the membrane was blocked with 3%
casein in TBST (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween-20) for 1
hour at room temperature. After washing, the membrane was incubated with
the hybridoma supernatant diluted 1/1000 in TBST, and incubated 1 hour
under gentle agitation. After extensive washing with TBST, the membrane
was incubated with a 115000 dilution of horse-radish peroxidase
conjugated goat anti-mouse IgG (Jackson #115-035-164) secondary antibody
for 1 hour. After washing, the membrane was developed with a
chemiluminescent substrate according to the manufacturer's
recommendations (Pierce #34080).

[0172]FIG. 5 shows that all mAbs detected a single band of a molecular
weight of about 100 KDa in LNCaP cell membrane (lane 1) and not in the
PC-3 cell membrane (lane 2). The fact that the antibodies detected a band
from a reducing and denaturing gel also confirm that they recognise a
linear amino acid sequence of PSMA as opposed to a conformational
epitope.

Immunohistochemical Staining of Prostate Cancer Tissue

[0173]Immunohistochemical staining was performed on paraffin embedded
section from prostate cancer. After deparafinization and rehydration
through graded alcohol, endogenous peroxidase was inactivated by treating
sections with 3% H2O2 for 20 min at RT. Non specific binding
was blocked with 5% normal goat serum (NGS) in 0.01 M phosphate buffered
saline pH 7.4; 0.05% Triton (PBS-T) for 30 min at RT before adding
primary antibodies diluted in PBS-T; 2% NGS overnight at RT. 8H12 was
used as a tissue culture supernatant diluted 1:5. Mouse IgG with an
irrelevant specificity was used as a negative control at a concentration
of 2 μg/ml. After washing, binding of primary antibody to tissue
sections was detected by sequential addition followed by washing of goat
anti-mouse Ig heavy+light chain polyclonal antibody (ICN) at 1:100, a
complex of horse radish peroxidase (HRP, 5 μg/ml) and a mouse
monoclonal antibody engineered to have dual specificity for goat antibody
and HRP (1/30), and DAB (0.06%); 0.01% H2O2 all diluted in
PBS-T; 2% NGS. Sections were washed in tap water, counterstained with
hematoxylin and rinsed in tap water. Sections were then dehydrated and
mounted in Permount® (Sigma). A pathologist evaluated all
immunohistochemical sections in a blinded fashion.

[0175]Staining of the benign prostatic glands, composed of prostatic
acinar cells and underlying basal cells, show that the basal cells are
PSMA negative, whereas the acinar cells are PSMA positive, mainly at the
luminal aspect of the plasma membrane (FIGS. 6B, C and D). 8H12 shows
moderate staining of PSMA in well differentiated prostate cancer, i.e.
Gleason 3+3=6. Weaker cytoplasmic staining is also seen.

In Vivo Biodistribution of Labeled Anti-PSMA mAbs

[0176]Purification of mAb: Cells were grown in Iscove's medium, 20% FCS,
IL-6 (1 mg/ml), and antibiotics using T175 flasks. After reaching
confluence, cells were removed by centrifugation. The medium was
precipitated with saturated ammonium sulfate (final concentration=45%)
overnight at 4° C. The solution was centrifuged and the
supernatant discarded. The precipitate was resuspended in PBS pH 7.4 and
further dialyzed against PBS at 4° C. A 5 ml protein G column
(Amersham) was equilibrated with 20 mM NaH2PO4 pH 7.0 and the
Ab solution was then passed through using a syringe barrel. The column
was washed with 20 mM NaH2PO4 pH 7.0 and finally elution was
done using Pierce's ImmunoPure Gentle Ag/Ab Buffer. Fractions containing
the Ab were combined and buffer exchanged into PBS using Amicon
Centriplus filtration devices.

[0177]Labelling of mAbs: 100 ug mAb were labelled by the method of
chloramine T (Bioconjugate Techniques (1996) Elsevier Science (USA)) by
mixing about 10 mCi NaI125 and five fold antibody molar equivalent
of chloramine T in a total volume of 135 ul. After 30 seconds, the
reaction was quenched with 100 ul sodium meta-bisulfite at a
concentration of 2.6 mg/ml. Free I125 was removed by gel filtration
of the antibody solution in a sodium phosphate buffer containing 0.1%
BSA. 85% to 92% of the radioactive iodine was associated with the
antibody, as assessed by HPTLC.

In Vivo Biodistribution of Labelled Anti-PSMA Mabs

[0178]In vivo targeting potential of the I125-8H12 and I125-29B4
was assessed in nude mice bearing LNCaP and/or PC3 tumors. Nude mice were
injected subcutaneously in the flank with 0.5×106 trypsinized
LNCaP cells and/or in the other flank with PC-3 cells in a volume of 200
ul PBS containing 50% Matrigel (Becton Dickinson). 1 month after the cell
injection, the mice were administered, by tail vein injection, 2 or 20 ug
of the mentioned labelled mAb at a specific activity of ˜2 uCi/ug.
After 24 or 48 hours post-injection, the mice were sacrificed and the
tumors and major organs were recovered and cleaned from blood. A blood
sample was also obtained at the time of sacrifice. The blood and tissue
samples were weighted and counted for radioactivity incorporation in a
gamma counter.

[0179]The relative activity of the tissue (cpm) was expressed per mg of
tissue. For mice bearing both LNCaP and PC-3 tumors, the ratio of the
relative activity of LNCaP/PC-3 tumor was calculated. For comparison of
mAb uptake between mice, relative tissue activity was first normalized to
blood to account for difference in the efficiency of injection, and then
the ratio of the relative activity of LNCaP tumor over non tumor tissue
was calculated.

[0180]FIG. 7 shows the LNCaP retention potential of the labeled Ab over
normal tissue 48 hrs after an injection. The LNCaP tumor retained the
labelled 8H12 antibody between 2.7 and 6.5 times better than the various
tissues tested. The tissue retention was comparable at 24 h
post-injection, indicating a complete bio-distribution of the mAb in a
minimum of 24 h. These results indicate a significant concentration of
8H12 in LNCaP tumor compared to major organs.

[0181]The selectivity of the 8H12 and 29B4 for LNCaP tumor compared to
PC-3 tumor was also measured in mice bearing both type of cells. Table 3
shows that Zug of the labelled 81-112 resulted in the concentration of
the mAb 4.3 times higher than in the PC-3 tumor.

[0182]While the invention has been described in connection with specific
embodiments thereof, it will be understood that it is capable of further
modifications and this application is intended to cover any variations,
uses, or adaptations of the invention following, in general, the
principles of the invention and including such departures from the
present disclosure as come within known or customary practice within the
art to which the invention pertains and as may be applied to the
essential features hereinbefore set forth, and as follows in the scope of
the appended claims.